Along with the respect to life quality and the rise of environmental protection consciousness, at present, the trend that the biological insecticides are substituted for the traditional pesticides to prevent the ultimate accumulation in the food chain has become the mainstream, in which Bacillus thuringiensis is the most famous application in the biological insecticides, and is easily utilized and safe.
Bacillus thuringiensis, a Gram-positive rod bacterium, is an insectile pathogenic bacterium. B. thuringiensis will progress into the non-mitotic semi-stationary phase or differentiate to form the spore or the insecticidal crystal protein while lacking nutrient or staying in the worse environment. The insecticidal crystal protein produced from B. thuringiensis can inhibit the growth of some insect pests, but is not harmful to mammals and birds. Therefore, scientists have been isolated various insecticidal genes from B. thuringiensis, and developed as the recombinant genetic products.
The endotoxin gene of B. thuringiensis is located on the plasmid thereof, so as to proceed the generic engineering easily. In the early stage, the recombinant endotoxin genes almost are limited in the cloning of single gene fragment. Recently, the multiple endotoxin genes or the genes with large diversities, even the chimeric genes, are utilized so as to improve the insecticidal effect, enlarge the insecticidal area or modify the resistance of B. thuringiensis to the worse environment.
The parental relationships among various endotoxin proteins of B. thuringiensis are different because of the insecticidal crystal proteins produced from the divergent nucleotide sequences of plasmids thereof. The insecticidal targets also are different, which are classified as six groups (Hofte and Whiteley, 1989; Gill et al., 1992; Gleave et al., 1993; Lereclus et al., 1993; Shin et al., 1995; Kostichka et al., 1996). Among these literatures, Cry1 protein family has the insecticidal effect to Lepidoptera; Cry2 protein family shows the insecticidal effect to Lepidoptera and Diptera, or only has the insecticidal effect to Diptera; Cry3 protein family has the insecticidal effect to Coleoptera; and Cry4 protein family only has the insecticidal effect to Diptera. Cry5 protein family cannot form as crystal protein, wherein Lepidoptera and Coleoptera can be killed by some part of Cry5 proteins but cannot be killed by other part thereof. CytA protein does not have specific insecticidal scope; however, the cytolytic and hemolytic effects can be induced by CytA protein. The cry1 gene of B. thuringiensis encodes the longest amino acid sequence, and cytA gene thereof encodes the shortest one.
Taiwan Patent No. 385229 relates to novel biopesticidal compositions comprising an active insecticidal ingredient selected from insecticidal bacteria and viruses such as B. thuringiensis crystal protein or spores or mixtures thereof and baculoviruses such as nuclear polyhedrosis viruses, granulosis viruses and non-occluded viruses. Methods for producing the biopesticidal compositions and methods of controlling insects are also included within the scope of the invention. The differences between the Taiwan patent No. 385229 and present patent lie on the way to anti insect. It is the endotoxin produced by the gene fragments are used to anti pest, rather than the crystal protein or spores or mixtures thereof and baculoviruses.
U.S. Pat. No. 6,500,617 provides methods of obtaining pest resistance genes that are improved over naturally occurring genes for use in conferring upon plants resistance to pests. The methods involve the use of DNA shuffling of pest resistance genes to produce libraries of recombinant pest resistance genes, which are then screened to identify those that exhibit the improved property or properties of interest. The present invention uses a naturally method to obtain the anti insect fragments, while the U.S. Pat. No. 6,500,617 uses an method with recombinant DNA.
The U.S. Pat. No. 6,177,615 disclosed a novel synthetically-modified B. thuringiensis nucleic acid segments encoding delta-endotoxins having insecticidal activity against lepidopteran insects. Also disclosed are synthetic crystal proteins encoded by these novel nucleic acid sequences. The differences between the U.S. Pat. No. 6,177,615 and the present patent lie on which the anti insect fragments of B. thuringiensis in present invention are naturally existent not by artificial synthesize.
The U.S. Pat. No. 5,994,266 relates to a method for controlling a pest comprising exposing the pest to the pesticidal compositions. The differences between the U.S. Pat. No. 5,994,266 and the present patent lie on which the anti insect fragments of B. thuringiensis in present invention are naturally existent not by artificial synthesize.
Therefore, scientists are still exploring the microorganism with multiple endotoxin genes isolated by generic engineering or isolated by natural selection.
It is therefore attempted by the applicant to deal with the above situation encountered in the prior art.